The invention concerns a method for the photometric analysis of test elements with detection zones, said method being stable in relation to positioning tolerances of the detection zone.
The photometric analysis of test elements is one of the most common methods for rapidly determining the concentration of analytes in samples. Photometric analyses are generally carried out in the field of analytics, environmental analytics and above all in the field of medical diagnostics. Test elements that are analysed photometrically play an important role especially in the field of blood glucose diagnostics from capillary blood.
The general trend in performing analytical tests is to reduce the amounts of sample. This is often due to the fact that only small amounts of sample are present. For example in the field of blood sugar determination, the diabetic himself removes a drop of blood from the finger pad and a reduction in the amount of blood required for the test considerably increases the convenience. This is primarily due to the fact that it is possible to select a smaller depth of cut for the blood collection. A reduction in the amounts of sample is linked to a miniaturization of the test elements and in particular of the detection zones in which the actual reaction of the sample with the analyte takes place. Hence in order to carry out an exact photometric analysis it is necessary to precisely position the detection zone. Numerous test element holders are known in the prior art which allow a quite accurate relative positioning of the detection zone and measurement optics. The European Patent EP-B-0 618 443 (corresponding to U.S. Pat. No. 5,424,035) is mentioned here as an example in which the detection zones of the test element are positioned laterally as well as vertically relative to the measurement optics. The document EP-A-0 319 922 is also mentioned as representative of many other analytical systems which carry out a positioning of test strips. However, the systems known in the prior art operate with detection zones which exceed 5 mmxc3x975 mm. Consequently a positioning in the manner known in the prior art is relatively unproblematic. However, if the lateral dimensions of the detection zone are reduced, great efforts have to be made in the manufacture of the test elements as well as in the positioning within an analytical system in order to obtain reliable analytical results. The object of the present invention was to propose a method and a device of the photometric analysis of test elements which also allow the reliable analysis of test elements with small detection zones.
This object is achieved by a method for the photometric analysis of test elements in which the test element is placed in a holder in which its detection zone is positioned such that it is disposed relative to an illumination unit with at least two light sources. After the detection zone has been contacted with the sample to be analysed, a first light source is activated in order to irradiate the detection zone. Light reflected from the detection zone or transmitted through the detection zone is detected as a first detection signal. Afterwards a second light source is activated which irradiates a second area of the detection zone which is offset from the first area in the direction of the expected positioning tolerance. The light reflected from this area of transmitted through this area is detected as the second detection signal. The detection signals obtained from the two areas are compared and examined to determine which of the signals was obtained by illumination of an area situated completely in the detection zone. A corresponding detection signal is selected and analysed to determine the analyte concentration in a basically known manner.
A further subject matter of the present invention is a method which can rapidly detect whether an adequate amount of sample liquid has been added to the test element or the detection zone and reports this back to the operator.
In the field of analysis with test elements it is a considerable gain in convenience when the time required to apply sample liquid to a test element can be reduced and when there is also a feedback about whether the applied amount of sample is sufficient. In the case of test elements which utilize an electrochemical principle, it has been common practice for some time to indicate to the user when sample liquid has penetrated into the analytical zone so that he can stop adding sample. In the case of test elements based on electrochemistry this detection can be achieved in a simple manner by a conductivity measurement. However, in the field of optical measurement of test elements such a rapid application detection cannot easily be accomplished. Methods are for example described in the documents U.S. Pat. No. 4,109,261 and EP-A-0 256 806 in which a sample application is detected by the optical system which carries out the measurement and is used as a start signal for awaiting an incubation period. However, in these described methods the user receives no feedback about whether he has applied sufficient sample liquid and can stop further addition. Furthermore optical systems are used in the described methods which detect colour formation in the detection zone of the test element. As shown in FIG. 7 of this application, several seconds pass between contacting the detection zone with sample liquid and the start of colour formation. If for example in the case of a blood sugar test the user has to hold his finger on the test element until a colour reaction is initiated this would be felt by him to be a constraint on his personal freedom. There is therefore a need to reduce the time for the addition or until the detection of the sample liquid. A blood sugar measuring instrument is described for example in EP-A-0 166 876 in which sample application is detected but it does not allow a check of whether the sample quantity is adequate.
Hence a method is proposed as part of the present invention which allows a rapid detection of sample application on a test element. The method according to the invention for the photometric analysis of test elements comprises the following steps:
irradiation of a control area of the detection zone,
transporting sample liquid to the detection zone in such a manner that a first zone of the detection zone comes in contact with the sample liquid earlier than a second zone which is at a lateral distance from the first zone
monitoring the radiation reflected from the control area or transmitted through the control area,
detecting presence of sample liquid in the control area as a result of a change of the reflected or transmitted radiation
irradiating a detection area of the detection zone
detecting irradiation which has been reflected from the detection area or transmitted through the detection area
analysing the detected radiation in order to determine the concentration of an analyte in the sample liquid,
characterized in that
when the presence of sample liquid is detected in the control area, a signal is emitted so that the supply of sample liquid can be terminated.
In the following the method according to the invention for compensating positioning tolerances is described first.
A device with which the method according to the invention can be carried out has a holder in which a test element is positioned relative to an illumination unit which is also part of the device. Holders that are known in the prior art and especially the holder described in EP-B-0 618 443 can be used in the following invention. Despite the inventive procedure which stabilizes the analysis in relation to positioning tolerances, it is desirable that the holder already results in a positioning of the detection zone that is as accurate as possible. However, the positioning tolerances cannot be reduced below a certain threshold which becomes clearer when one considers the individual factors that impact on the positioning tolerances. The analytical system already results in a number of tolerances for a relative positioning of the holder and illumination unit. Further tolerances are caused by the positioning of the test element within the holder since a certain clearance between the test element and holder is necessary so that the user can insert the test element in the holder. Furthermore the test element itself is subject to certain manufacturing tolerances which relate in particular to the width of the test element. Finally tolerances also occur with respect to the position of the detection zone relative to the test element and the carrier material of the test element. Especially in capillary gap test elements the detection zone may be considerably displaced in the direction of the width of the test element which is described below in more detail.
The device according to the invention for photometric analysis additionally comprises an illumination unit with at least a first and a second light source. These light sources are used to illuminate adjacent areas of the detection zone. The light sources are arranged such that the centres of the illuminated areas on the detection zone are displaced relative to one another in the direction of an expected positioning tolerance. This can for example be achieved in that the light sources themselves are displaced relative to one another and radiate essentially perpendicularly onto the detection zone. However, the light sources can also be arranged such that they obliquely irradiate the detection zone and the distance between the irradiated areas is essentially achieved by the slant of the light sources relative to the perpendicular relative to the detection zone. In principle the light sources known in the prior art for diagnostic detection systems are suitable as light sources. Light-emitting diodes are preferred as these have a relatively low energy consumption and yield a relatively narrow band spectrum. Miniaturized light-emitting diodes which can be mounted on a semiconductor circuit board are particularly preferred. Such light-emitting diodes are obtainable in sizes which result in light spots of a suitable size on the detection zone without interposing an optical system. However, in the framework of the present invention it is preferable to use an optical system comprising lenses and optionally apertures in addition to the light sources. It is advantageous to adapt the shape of the irradiated areas to the shape of the detection zone and to the expected tolerances. In the case of a rectangular detection zone it is for example advantageous to illuminate oval areas or even rectangular areas. This can for example be achieved by suitable lenses or apertures.
The present invention also envisages using more than two light sources. If a particularly large positioning tolerance is expected in one direction of the detection zone, it is possible for example to use three light sources which illuminate areas on the detection zone which are displaced in the direction of the expected positioning tolerance. The present invention is also suitable for reducing the impact of positioning tolerances in more than one direction. For example four light sources can be used which illuminate four areas that are arranged in the shape of a square or rectangle. Such an arrangement can prevent measurement errors due to positioning inaccuracies in the direction of the one side of the square as well as in the perpendicular direction.
In a method for photometric analysis of test elements a sample is contacted with the detection zone of the test element. The contacting is understood either as a direct application of the samples on the detection zone or a transport of the sample into the detection zone. The latter is in particular the case for capillary gap test elements in which the sample (usually capillary blood from the finger pad) is guided to the capillary gap and is transported through this into the detection zone. Embodiments are also conceivable in which a solid sample is applied to a fleece by for example rubbing a fleece on an object and the sample is subsequently transported by means of an auxiliary fluid from the fleece to the detection zone as is for example the case in some rapid drug tests. In addition there are chromatographic test strips in which the sample is transported into the detection zone by means of absorbent materials. Hence the aforementioned shows that samples within the sense of the invention are primarily sample liquids such as blood but solid sample materials can also be used.
Sample that has been brought into contact with the detection zone leads to a detectable change of the detection zone in test elements. The generation of detectable changes as a result of a reaction of an analyte contained in the sample with a reagent system is well-known in the prior art and thus does not have to be further elucidated here. However, it is relevant for the present invention that the detectable change can be adequately detected with the light sources and detector that are used. This is the case when a colour is formed in the detection zone which absorbs the radiation emitted by the light sources and thus attenuates the transmitted or reflected light. However, the converse case is also possible in which a dye whose presence is detected photometrically is destroyed by the reaction of the reagent system with an analyte and the actual measured signal is a decrease in the attenuation of the transmitted or reflected light. The term light or light source should be understood in the scope of the invention to encompass wavelength ranges that can be used by optical arrangements i.e. UV and IR in addition to the visual range.
The well-known detectors in the prior art and especially semiconductor detectors can be used for the present invention. It is important to select the detector or detectors such that the radiation reflected from the detection zone or transmitted through the detection zone leads to a signal when the detector is illuminated with this radiation. Detectors which have their sensitivity maximum in the range of the reflected or transmitted radiation can be used advantageously. Optionally it is also possible to use filters which allow the measurement radiation to pass selectively in order to make the detection more stable towards the effects of interfering light.
In order to carry out the method according to the invention a first light source of the illumination unit is firstly activated to irradiate a first area of the detection zone and the signal present at the detector during the activation is recorded as a first detection signal. Subsequently the first light source is deactivated and the second light source is activated to irradiate a second area and the signal present at the detector is recorded as the second detection signal. Electric circuits and methods for activating light sources, especially light-emitting diodes are sufficiently well-known in the prior art. Reference is made by way of example to the document U.S. Pat. No. 5,463,467 in which an activation of light-emitting diodes is described for carrying out photometric analyses. The present invention also envisages activating light sources by means of signals which enable the influence of foreign light to be eliminated from the detected signal as described for example in the said document U.S. Pat. No. 5,463,467.
An important step in the method according to the invention is to compare the detected signals and determine which of the signals or whether both signals have been obtained by illumination of an area which lies completely on the detection zone. This determination can be carried out in various ways. If the analyte results in a colour forming reaction in the detection zone, the first and second detection signal can be compared and the light signal that has been more greatly attenuated is selected as lying completely on the detection zone. If in contrast a reaction is carried out in which colour is degraded by the analyte, the signal which has been subjected to the least attenuation is selected as the one which is located completely on the detection zone. These two procedures can be carried out without having to carry out a measurement on the detection zone before reaction with the analyte in order to determine a blank value. However, according to the invention it is preferred that first measurements are carried out on the detection zone with a first and second light source before carrying out the reaction with the sample. The detection signals determined in this manner can be used as a basis for determining which signal results from an irradiated area that is located completely within the detection zone and as a base value (so-called blank value) for determining the analyte concentration. If measurements have been carried out with the light sources on a test element that has not yet reacted, then the inventive determination step can be advantageously carried out by determining the signal amplitude before and after reaction of the detection zone with the sample and selecting that light source for the determination of the analyte concentration which yielded the larger signal amplitude.
The selection of at least one of the two light sources as suitable for determining the analyte concentration can also be carried out without having to contact the detection zone with the sample. This can for example be achieved when the detection zone is composed of a light material and the test element has a dark colour in the area bordering the detection zone. If measurements are carried out on such a test element with a first and second light source and the measurements are compared, the light source from which the higher reflectance signal was obtained can be selected as being suitable for determining the analyte concentration. It is additionally possible to make the detection zone at least partially transparent whereas the area of the test element bordering the detection zone is essentially impermeable to light. If a transmission measurement is now carried out, the light source can be selected with which a higher transmission was obtained. If reference was made above to a higher or lower signal, this can mean a xe2x80x9ccrude signalxe2x80x9d which is present at the detector when the light sources are activated. The crude signals are advantageously corrected before comparing them i.e. the illumination geometry and the variation of intensity from light source to light source are taken into account. This can for example be achieved by standardizing the signals on the basis of blank values (forming a ratio between the actual signal and the signal for an unreacted test element or standard).
The present invention additionally concerns a method for the photometric analysis of a test element in which sample application is detected. In this method as so-called control area of the detection zone of a test element is firstly irradiated and the radiation reflected from this control area or transmitted through the control area is monitored. In this connection monitoring means that the reflected or transmitted radiation is measured at intervals. Usually measurements are carried out at intervals of fractions of a second. The time intervals are selected in practice such that the time interval between two measurements is less than the time delay which can be tolerated for the detection of a change in the measured value. During the monitoring of the control area, a sample liquid is supplied to the detection zone and this is carried out in such a manner that a first zone of the detection zone comes into contact with the sample liquid earlier than a second zone which is laterally spaced from the first zone. In this connection the term lateral refers to the plane of the detection zone. Hence there is a spacing between the first and the second zone when the detection zone is observed from the upper side. Such a transport of sample liquid to a detection zone occurs for example in chromatographic test strips in which the sample or a reaction solution of the sample penetrates from one side into the detection zone and laterally migrates through the detection zone. Such a supply of sample liquid also occurs when a capillary gap test element is used in which the capillary gap and detection zone are disposed in the same plane. This can for example be achieved in such a manner that the capillary gap ends at an edge of the detection zone and sample liquid from the capillary gap penetrates from this site of contact into the detection zone or the capillary gap can be located below the detection zone as shown in FIG. 1. In the said cases the sample liquid comes into contact with the first zone of the detection zone earlier than with a second zone. In FIG. 6 this is indicated by the zones Z1 and Z2. The sample liquid penetrates via a capillary gap which is located below the detection zone (10) into the detection zone in the direction of the arrow. In this case the zone Z1 comes into contact with the sample liquid earlier than zone Z2.
In the method according to the invention a sample application is detected on the basis of a change in the reflected or transmitted radiation in the control area. In this connection a change means that a signal change occurs during the monitoring i.e. the transmission or reflection signals obtained from the unreacted control area of the detection zone are different from those obtained from a control area which is moistened thoroughly with sample liquid or which has reacted with sample liquid.
In order to analyse the test element, at least one detection area of the detection zone is irradiated and the radiation that is reflected from this area or transmitted through the detection area is detected and analysed in order to determine the concentration of an analyte in the sample liquid. Details on the detection and analysis have already been described above for an inventive method for compensating positioning tolerances.
Monitoring the control area enables an earlier detection of the presence of sample liquid in the control area than when measuring in the detection area. When liquid is detected a signal can be emitted which terminates supply of sample liquid. The signal can be an optical and/or an acoustic signal. In the case of a capillary gap test element this for example means that blood supply to the capillary gap can be terminated. Consequently it is possible to prevent unnecessary supply of further sample liquid and thus to reduce the required amount of sample and on the other hand this procedure shortens the time required to supply sample liquid which is convenient for the user. Nevertheless the method according to the invention still ensures that the detection area or detection areas of the detection zone are adequately supplied with sample liquid. For this purpose it is advantageous when the detection area is nearer than the control area to the first zone which firstly comes into contact with the sample liquid. Thus the test element is wetted with sample liquid in the area of the detection zone earlier than in the area of the control zone and when sample liquid is present in the area of the control zone it is ensured that the detection zone is also supplied with sample liquid. According to the invention it is also preferred that the control area (area A in FIG. 6) is irradiated with radiation that is absorbed by the sample liquid itself. Even if the sample liquid does not itself absorb radiation or only partially absorbs radiation there is usually a decrease in the reflected or transmitted radiation when the control zone is moistened. As a result the presence of sample liquid can already be determined before a reaction with reagents has taken place in the detection zone. FIG. 7 shows that a decrease in reflection in area A of the detection zone shown in FIG. 6 is already found in the time interval II. This is due to the fact that a thorough moistening of the control area A can be very rapidly detected by a 880 nm light-emitting diode. Detection of a wetting of this area on the basis of a colour formed in the detection zone would not have been possible until time interval IV. In the method according to the invention in which sample application is detected, the control area is preferably on the detection zone. This not only reduces the time until detection but also allows a compact construction of such an instrument in which the optical components can be in close vicinity to one another.